Kit and method for determining redox status in urine

ABSTRACT

A method and corresponding kit for determining the redox status of an organism is provided. The method comprises performing a calorimetric assay of a urine sample by mixing the sample with at least one member of a group of disclosed reagents and an acid and, upon stable color formation, comparing the resulting color to a color scale to indirectly determine the oxidative load, or stress, experienced by that organism. The corresponding kit comprises at least one of the reagents described above, an acid, and a color scale.

[0001] This is a continuation-in-part of international patentapplication PCT/EP00/13187 filed Dec. 22, 2000, from which priority isclaimed in the present application.

[0002] This invention relates to a kit and method for determining redoxstatus in urine. The invention relates in particular to such a kit andsuch a method in which the determination of redox status can be carriedout extremely easily on the basis of the color reaction with a reagentselected from the group of indole derivatives by visual colorcomparison, i.e., with the naked eye.

BACKGROUND TO THE INVENTION

[0003] The human body is exposed to a large number of loads causing freeradicals to be produced in the cells of the body, particularly under thepresent environmental conditions. These loads include, for example,cigarette smoke, car exhaust gases, UV radiation, and various othernoxious substances. The free radicals produced, which also includeoxygen free radicals, form pathogenic products of degradation, which areable to cause sustained damage to various components within the cells ofthe body cell in a wide variety of subsequent oxidative reactions. Theconsequences of this damage include, for example, premature aging, butalso serious diseases such as diabetes or cancer.

[0004] In order to neutralize, i.e., bind free radicals, which alsooccur to a limited degree under ideal environmental conditions, thehuman body has developed a number of systems with an anti-oxidativesystem. However, these systems are frequently overloaded, among otherthings, because of lifestyles and the particular environmental situationof the human being, so that free radicals in the body cannot be renderedsufficiently harmless. In such cases, it is necessary to stimulate orsupport the body's own anti-oxidative systems by eating special foodsupplements (micro-nutrient preparations from vitamins and traceelements). In such a treatment, the quantity of drugs administered orsupplements taken should be adapted to the actual loading of the bodywith free radicals.

[0005] Ideally, the determination of the load exerted by free radicalsshould be carried out by the patient himself/herself without majorexpenditure, quickly, and at low cost. Such a determination maytherefore be carried out on the basis of urine samples in particular.

SUMMARY OF THE INVENTION

[0006] The fundamental objective of this invention is therefore todetermine redox status in urine by simple means, i.e., by the naked eye.“Determination of redox status” is defined in this connection asobtaining a quantitative, semi-quantitative, or qualitative statement onthe concentration and presence of free radicals in the human body, i.e.,on the oxidative loading of the organism. The malonic dialdehyde in theurine serves as an indicator substance for this statement, but otherrelevant substances can also be recorded, as appropriate.

[0007] This objective is achieved according to the invention by a kitfor determining redox status in urine, comprising:

[0008] (a) at least one reagent selected from the group of compoundswith the general formula (I)

[0009]  wherein R¹ is H or C₁-C₁₀-alkyl;

[0010]  and wherein R² is independently selected from the groupconsisting of H; C₁-C₁₀-alkyl; NH₂; NHR; NR₂; NHCOR; OH; OR; OCOR; SH;SR; F; Cl; Br; CF₃; or CCl₃, and wherein each R is selected from thegroup consisting of C₁-C₆-alkyl;

[0011]  and wherein R³ and R⁴ are independently selected from the groupconsisting of H; C₁-C₁₀-alkyl; NH₂; NHR; NR₂; NHCOR; OH; OR; OCOR; SH;SR; F; Cl; Br; CF3; CCl₃; C₆-C₁₄-aryl; C₆-C₁₄-aryl, wherein the arylgroup is substituted by one or more substituents selected from the groupconsisting of C₁-C₆-alkyl, NH2, NHR, NR₂, NHCOR, OH, OR, OCOR, SH, SR,F, Cl, Br, CF₃ and CCl₃, and wherein each R is selected from the groupconsisting of C₁-C₆-alkyl;

[0012] (b) at least one acid; and

[0013] (c) a color scale for the visual color comparison, where acertain redox status of the urine to be analyzed is assigned toindividual colors on the scale.

[0014] According to a further embodiment of the invention, a method isalso provided for determining redox status in urine, comprising thefollowing stages:

[0015] (a) Mixing of a urine sample to be analyzed with

[0016] (i) at least one reagent selected from the group of compoundswith the general formula (I)

[0017]  wherein R¹ is H or C₁-C₁₀-alkyl;

[0018]  and wherein R² is independently selected from the groupconsisting of H; C₁-C₁₀-alkyl; NH₂; NHR; NR₂; NHCOR; OH; OR; OCOR; SH;SR; F; Cl; Br; CF₃; or CCl₃, and wherein each R is selected from thegroup consisting of C₁-C₆-alkyl;

[0019]  and wherein R³ and R⁴ are independently selected from the groupconsisting of H; C₁-C₁₀-alkyl; NH₂; NHR; NR₂; NHCOR; OH; OR; OCOR; SH;SR; F; Cl; Br; CF₃; CCl₃; C₆-C₁₄-aryl; C₆-C₁₄-aryl, wherein the arylgroup is substituted by one or more substituents selected from the groupconsisting of C₁-C₆-alkyl, NH2, NHR, NR₂, NHCOR, OH, OR, OCOR, SH, SR,F, Cl, Br, CF₃ and CCl₃, and wherein each R is selected from the groupconsisting of C₁-C₆-alkyl;

[0020] (ii) at least one acid; and

[0021] (b) after a stable coloration of the sample has been achieved,visual comparison of the coloration of the sample with a color scale, aspecific redox status of the urine to be examined being assigned to theindividual colors of the scale.

[0022] The kit and the method according to the invention are based on acolor reaction, which shows the reagent having the formula (I) underacid conditions when mixed with the urine sample to be analyzed. Thiscolor reaction results in the formation of a colored dye characteristicof the initial reagent, the intensity of the coloration depending on theoxidative loading of the organism. In this case, the heavier this load,i.e., the higher the concentration of free radicals in the body, themore intensive the coloration. In general, the color of the sample thatcan be perceived with the naked eye is also influenced by the color ofthe urine analyzed.

[0023] This color reaction is initiated in particular by the presence ofmalonic dialdehyde in the urine analyzed. Malonic dialdehyde is aproduct of degradation of certain hydroperoxides, which are formed bythe oxidation of unsaturated fatty acids of the cell membrane in theorganism. Since oxidative stress results in intensified lipid oxidationof the cell, it also causes an increase in the concentration of malonicdialdehyde in the urine. This increase can be determined by means of thekit and method according to the invention, so that countermeasures canthen be taken, depending in particular on the intensity of the load,e.g., measures such as strengthening the anti-oxidative defense bytaking suitable vitamins and trace elements and/or altering one'slifestyles (smoking, diet, living/working environment).

[0024] The redox status is determined by means of the kits according tothe invention, and by the method according to the invention, by mixingtogether the reagent, the acid, and the urine sample at roomtemperature, whereupon the coloration stabilizes, depending inparticular on the intensity of the oxidative load. The color reaction isgenerally completed within 15 to 60 minutes, and in preferredembodiments within 15 to 30 minutes. Afterwards, i.e., as soon as theintensity of the coloration stops increasing, this intensity isdetermined visually, i.e., by the naked eye, by comparison with a colorscale. In this case, the color scale consists of a number of discretecolor elements, which are arranged according to their intensity andwhose colors correspond to the coloration of the sample solution aftertermination of the color reaction, according to redox status.

BRIEF DESCRIPTION OF THE DRAWING

[0025] FIGS. 1 to 3 show preferred embodiments of two- or multi-chambersystems that can be used according to the invention.

[0026] FIGS. 4 to 6 show preferred embodiments of test strips that canbe used according to the invention.

DESCRIPTION OF PREFERRED EMBODIMENTS

[0027] The reagents with the general formula (I) are indole derivatives.In formula (I), R¹, R², R³, and R⁴ denote, among other things, an alkylgroup with 1 to 10 carbon atoms. Examples of such alkyl groups aremethyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl,tert-butyl, pentyl, and hexyl. Alkyl groups with 1 to 6 carbon atoms arepreferred, and those with 1 to 4 carbon atoms are particularlypreferred. A particularly preferred alkyl group is the methyl group.

[0028] In formula (I), R³ and R⁴ may also denote an aryl group with 6 to14, preferably 6 to 10 carbon atoms, among other things. This aryl groupmay, as appropriate, be substituted by one or more substituents selectedfrom the group consisting of C₁-C₆-alkyl, NH₂, NHR, NR₂, NHCOR, OH, OR,OCOR, SH, SR, F, Cl, Br, CF₃, and CCl₃, in which R denotes C₁-C₆ alkyl,and where several R groups may optionally be selected independently. Aparticularly preferred aryl group is phenyl.

[0029] In formula (I), R², R³, and R⁴ may also denote NH₂, NHR, NR₂,NHCOR, OH, OR, OCOR, SH, SR, F, Cl, Br, CF₃, or CCl₃, where R denotesC₁-C₆-alkyl and where several R groups may optionally be selectedindependently. These groups are, in particular, substituents with apositive mesomeric effect. A particularly preferred group of this kindis the methoxy group.

[0030] Preferred reagents with the general formula (I) are: 2-methylindole, 1,2-dimethyl indole, 1-methyl indole, 2-phenyl indole, and1-methyl-2-phenyl indole. These reagents are particularly preferred, fortheir stability or the long-term storage stability of their solutionsand/or for their availability in (highly) pure form.

[0031] According to the invention, an acid, which may be inorganic ororganic, is used. Suitable acids include, for example, hydrochloricacid, sulphuric acid, phosphoric acid, methane sulphonic acid, citricacid, oxalic acid, trifluoroacetic acid, or trichloroacetic acid. Theuse of hydrochloric acid is particularly preferred.

[0032] The reagent and/or acid may be used in the form of a solution ina suitable solvent. In principle, all solvents, which are miscible withwater and do not have a highly toxic action, are suitable. Loweralcohols, such as methanol, ethanol, propanol, isopropanol, or butanolare particularly suitable, but in closed systems acetonitrile,tetrahydrofuran, or hexamethyl phosphoric acid triamide may also besuitable. Ethanol and isopropanol are preferred, the latter beingparticularly preferred.

[0033] When the reagent is used in the form of a solution, itsconcentration in it is generally 0.1 to 50 mmol/l, preferably 5 to 15mmol/l, and more preferably 7 to 12 mmol/l.

[0034] When the acid is used in the form of a solution, itsconcentration in it is generally 0.1 to 5 mol/l, preferably 0.5 to 1.5mol/l, and more preferably 0.9 to 1.2 mol/l.

[0035] A solution of hydrochloric acid in methanol, ethanol, orisopropanol is used in preference as an acid, a solution of hydrochloricacid in isopropanol being particularly preferred.

[0036] The color scale or color matrix can be set up by preparing modelurine solutions with various known concentrations of malonic dialdehydeand various intensities of yellow coloration and carrying out theabove-described method on these solutions. The end point colors thusobtained are then reproduced in the form of a one-dimensional scale or atwo-dimensional matrix.

[0037] Since the coloration of the urine sample analyzed generally alsodepends on the color of the urine to be analyzed, i.e., its yellowcoloration of varying intensity, it is appropriate to supply differentcolor scales for the individual initial urine colors. These scales may,in particular, be arranged in the form of a color matrix, the first lineof the matrix containing the urine colors considered, whosecorrespondence is established initially by comparison with the untreatedurine. The color comparison with the sample after the end of the colorreaction is then carried out inside the color matrix column, whichestablished by the correspondence of the color of the initial urine.

[0038] With regard to the cost of determining the redox status using thekit and method according to the invention, the components required forthis, i.e., the reagent and the acid, are supplied and used in such aform that their portioning or handling are simplified as much aspossible. In this connection, however, consideration must be given tothe fact that solutions of the reagent exhibit a low storage stabilityunder acid conditions, so that the reagent and the acid cannot bebrought into contact with each other until shortly before the actualdetermination is carried out.

[0039] According to a particular embodiment of the invention, thereagent or a solution of it is therefore enclosed in a container,thereby separating it from the acid. This container can easily bedestroyed or opened by deliberate mechanical action and is arranged sothat the reagent comes directly into contact with the acid and/or with asolution of it and/or with the urine sample due to the destruction oropening of the container. The container mentioned may, for example, be aglass ampoule. This glass ampoule may, in turn, be arranged inside avessel, which contains the acid or a solution of it, and to which theurine sample is fed in order to carry out the determination. The colorreaction can then be initiated by breaking the glass ampoule inside thevessel so that the content of the ampoule and the vessel mix together.

[0040] Accordingly, the acid, instead of the reagent, can also beenclosed in a separate container, which is arranged as described aboveand is opened or destroyed correspondingly.

[0041] A further embodiment of the two-chamber system that can be usedaccording to the invention is represented diagrammatically in FIG. 1.The system shown in that figure consists of two vessels 11 and 16, eachof which is provided with screw threads 12 and 14 respectively, wherebythey can be connected together. During this connection, a membrane 15,with which vessel 16 is sealed, pierces a pointed opening 13 of vessel11. In vessel 16, there is a reaction component 17 (reagent or acid),while the other component (acid or reagent) is contained in vessel 11,and does not reach vessel 16 until after membrane 15 has pierced it.

[0042] Vessel 11 may advantageously be designed as a plastic bottle,which can be compressed for transferring its content into vessel 16. Theurine sample to be analyzed may also be sucked into vessel 11, in thiscase, before it is transferred together with one of the reactioncomponents into vessel 16.

[0043] A two-chamber system that can be used according to the inventionmay also be designed as shown diagrammatically in FIG. 2. The systemshowed in this figure consists of two vessels 23 and 27, which areprovided with screw threads 25 and 26 and can therefore be tightlyconnected to each other. In vessel 23, a chamber 22, which contains oneof the reaction components (reagent or acid), is formed by a membrane24. Membrane 24 is pierced by pressing pin 21 inwards so that thereaction component contained in chamber 22 comes into contact with theother component 28 (acid or reagent) contained in vessel 27. The urinesample to be analyzed may advantageously be fed into vessel 27 beforemembrane 24 is pierced, but also after the reagent and acid are mixedtogether, as appropriate.

[0044] In a modification of the system as shown in FIG. 2, pin 21 mayalso be omitted if the threads and membrane are designed so that themembrane is torn apart when the vessels are screwed together.

[0045] A further advantageous variant of a multi-chamber system that canbe used according to the invention is shown in FIG. 3. This system alsoconsists of two vessels 34 and 37, which can be connected together byscrew threads 35 and 36. The reagent and the acid are herein containedin two chambers 32 and 33 designed to be connected to vessel 34, and arebrought into contact with each other by actuating piston 31. In thissystem, vessel 37 may advantageously be designed as a beaker useddirectly for receiving the urine sample.

[0046] With regard to the dosing and handling of the reagent, this canbe advantageously effected not only in pure form, but also by mixingwith a neutral carrier material, such as powder, a tablet, or on a solidcarrier (e.g., a plastic strip). The following may be considered neutralcarrier substances, given by way of examples: neutral alkali salts,e.g., sodium chloride, or inert carriers of higher density, e.g., SiO₂.The weight ratio of reagent to carrier substance is in this casegenerally 1:0 (pure reagent) to 1:1000, preferably 1:0 to 1:200.

[0047] To facilitate the determination of redox status, the kitaccording to the invention may contain a vessel for directly receivingthe excreted urine. This vessel may be designed, for example, in theform of a simple beaker. Furthermore, the kit according to the inventionmay contain a means of pipetting, for example. This may be used, inparticular, for transferring a measured quantity of the urine to beanalyzed from the aforementioned vessel for sampling to the vessel inwhich the color reaction is carried out. Commercially availabledisposable pipettes may be used, in particular, as means of pipetting.

[0048] Moreover, the kit, according to the invention, may contain ananalysis vessel in which the color reaction is carried out by mixingtogether the reagent, the acid, and the urine sample. According to aparticularly advantageous embodiment, the color scale may be connectedto the analysis vessel so that the sample solution and the individualcolor elements of the scale are located in the immediately spatialvicinity, so that the color comparison can be carried out immediately,i.e., at a glance.

[0049] According to a further advantageous embodiment of the invention,the reagent and the acid are both worked in solid form into a teststrip, which can be immersed directly into the sample to be analyzed. Inthis case, citric acid, oxalic acid, or trichloroacetic acid may be usedas the acid.

[0050] A simple embodiment of such a test strip is representeddiagrammatically in FIG. 4. In the case of the test strip 44 shown inthat figure, the acid is applied in two discrete areas, 41 and 43,between which there is an area 42, which contains the reagent. When thetest strip is immersed in the urine sample to be analyzed, the aciddiffuses into reagent area 42, where the color reaction takes place.Once this has finished, the color of this area is compared with thecolor scale.

[0051] A test strip that can be used according to the invention may alsobe designed in the form of a test cassette shown diagrammatically inFIG. 5. Cassette 52 contains a fleece strip to which the urine sample isapplied in the area of opening 53. The sample then spreads through thestrip in the direction indicated by arrow 51. The acid is applied inarea 54, and is absorbed by the solution on its way to area 55containing the reagent, so that the color reaction takes place in area55. The color comparison can then be carried out through a suitableopening.

[0052] According to the invention, a test strip may also be designed orused as shown diagrammatically in FIG. 6. Here the acid is contained inarea 63 and the reagent in area 62 of strip 61. Strip 61 is immersed inurine sample 65 to be analyzed, which is located at the bottom of vessel64. The sample solution then spreads up and out by capillary forces. Indoing so, it passes into area 63, where it absorbs the acid before thecolor reaction takes place in area 62.

EXAMPLES

[0053] This invention is explained in greater detail with reference tothe following examples:

Example 1 Determination of Redox Status

[0054] According to three different variants, the components (reagentand acid) indicated in the following Table 1 are each mixed in thequantities also indicated in Table 1 with 1 ml of fresh morning urine.The mixture thus obtained is shaken vigorously in a sealed vessel andallowed to stand. After 15 to 20 minutes, the coloration production iscompared to the color scale. TABLE 1 Variant Component CompositionQuantity 1 Reagent 100 mg of 2-methyl indole dissolved 1 ml in 100 ml ofisopropanol Acid 25% aqueous HCl solution 1 ml 2 Reagent 100 mg of2-methyl indole dissolved 1 ml in 50 ml of isopropanol Acid  25 ml of25% aqueous HCl solution, 1 ml mixed with 50 ml of isopropanol 3 Reagent100 mg of 2-methyl indole mixed 0.20 g with 20 g of sodium chloride Acid 25 ml of 25% aqueous HCl solution 2 ml mixed with 100 ml of isopropanol

Example 2 Testing of the Behavior of Special Reagents

[0055] The behavior of the following reagents A to E, that can be usedaccording to the invention, was examined in this example:

[0056] In this case, aqueous solutions of malonic dialdehyde were usedas the “model urine,” with concentrations of 10 μmol/l and 1.0 μmol/l,respectively. (Typical malonic dialdehyde concentrations in human urineare of the order of 0.5 to 5 μmol/l).

[0057] A solution of 100 mg of reagent (A to E) dissolved in 50 ml ofisopropanol was used as the reagent solution, and a mixture of 25 ml of25% aqueous HCl solution and 50 ml of isopropanol was used as the acidsolution.

[0058] The test was carried out by mixing 1 ml of the test solution with1 ml of reagent solution and 1 ml of acid solution. The mixture thusobtained was vigorously shaken in a sealed vessel and allowed to standat 22° C. The colorations that occurred were then determined once after15 to 20 minutes, then again after 60 minutes, and are indicated in thefollowing Table 2.

[0059] The individual colorations were in this case determined bycomparison with the HSKTM standard color tables and fans (“K” screenfans, 1993 Issue, Hostmann-Steinberg, Celle, sales through HKVdruckGmbH, Stuttgart), the color code (e.g. “45K”) and saturation orbrightness stage (e.g. 60%) at a grey stage of 0% being indicated inTable 2.

[0060] Reagents A and B give red color tones, reagent C gives ablue-grey color tone and reagents D and E give blue color tones. TABLE 2Malonic dialdehyde concentration 10 μmol/l 1.0 μmol/l Reaction timeReaction time Reagent 15 to 20 mins. 60 mins. 15 to 20 mins. 60 mins. A27K (100%) 27K (100%) 27K (100%) 27K (100%) B 31K/29K* (100%) 31K/29K*(100%) 31K/33K* (100%) 31K/33K* (100%) C 36K (100%) 82K (40%) 28K (100%) 1K (60%) D 45K (60%) 45K (100%) 26K (20%) 34K (60%) E 40K (80%) 43K(100%) 40K (10%) 40K (50%)

[0061] The following graduations of the reaction speed (in descendingorder) or the individual reagents were established on the basis of thesetests:

[0062] (a) at a malonic dialdehyde concentration of 10 μmol/l:

A=B=C>D>E

[0063] (b) at a malonic dialdehyde concentration of 1.0 μmol/l:

A>B>C>>D>E

[0064] In a similar test with variation of the acid used (according toExample 1, Variant 2; malonic dialdehyde concentration=10 μmol/l), thefollowing graduation of the reaction speed (in descending order) wasdetermined: hydrochloric acid>>trichloroacetic acid>oxalic acid>citricacid.

Example 3 Monitoring of Redox Status when Administering Micro-Nutrientswith an Oxidative Action

[0065] Starting from urine samples not subject to oxidative load, withyellow colorations of varying intensities (e.g., which may correspond tothe HKS™ reference colors 1K, 3K and 5K; see the data given in Example2), and using malonic dialdehyde dilution series, a color scale wasproduced for each initial urine color, its graduations being denoted inthe following by I (low oxidative load), II (medium oxidative load), andIII (high oxidative load).

[0066] A first-dimension color scale or “urine color control scale” isdeveloped to account for the varying yellow coloration of urine (using,e.g., the HKS™ reference colors). The “urine color control scale”provides a continuous or punctuated spectrum of yellow, representativeof the varying yellow colorations of urine. The “urine color controlscale” eliminates the potentially confounding effect that the initialcolor of a tested urine might have on the results achieved using themethods or kits of the invention. The “urine color control scale” isgenerated, for example, by collecting a number of urine samples from arepresentative range of human subjects, preferably a number of humansubjects that can provide a set of urine samples collectively ranging incolor from relatively pale to relatively intense yellow. Alternatively,the “urine color control scale” is generated by adding varyingquantities of a yellow dye to a solvent, preferably water (e.g., byserial dilution), in order to produce a series of solutions with varyingyellow intensities that approximately matches the various yellowcolorations of urine. Preferably, these dye solutions collectively rangein intensity from relatively pale to the relatively intense yellow.Alternatively, the “urine color control scale” is generated by usingyellow filters ranging in intensity from the relatively pale to therelatively intense yellow colors characteristic of urine.

[0067] A second-dimension color scale, or “reaction color scale,” isdeveloped from each member of the urine color control scale by holdingconstant the initial color intensity of a solution while varying theconcentration of MDA (malonic dialdehyde). Individually, asecond-dimension reaction color scale (each individual reaction colorscale being associated with a single member of the first-dimension, orurine color control, scale) accounts for the varying concentrations ofMDA in urine of a particular color intensity, with the range of MDAconcentrations being indicative of the range of low (I) to high (III)oxidative loads in an organism. Collectively, the reaction color scalesinclude the first-dimension urine color control scale, therebyaccounting for both the varying initial coloration of urines amenable totesting and the color of products resulting from reactions of varyingconcentrations of MDA.

[0068] The “reaction color scale” is generated, for example, by varyingthe concentration of MDA in aliquots of a single fluid of determinablecolor intensity, including a lack of coloration such as is found withwater, a preferred fluid. MDA of differing concentrations is generatedby any method known in the art. For example, a known MDA concentrationmay be diluted by, e.g, serial dilution. This results in a lineardilution series or a non-linear dilution series, such as serialdilutions that progressively reduce the MDA concentrations by 50%, 90%,or any other suitable increment. It will be recognized by those of skillin the art that the incremental variation need not be constant. One ofordinary skill in the art will be able to use any method known in theart to generate a series of varying MDA concentrations that correspondto the lowest to highest concentrations of relevant substances (i.e.,substances reactive with at least one each of the reagents and acidsdisclosed herein to yield colored products) found in human urine.Typically, the relevant substance is MDA itself, and the followingdescription is presented in terms of MDA. Alternatively, reaction colorscales are prepared, for example, using urine, recognizing that theoriginal concentration of MDA in the urine is unknown.

[0069] “Reaction color scales” can also be generated, for example, bypreparing a series of varying MDA concentrations in a MDA-free solvent,preferably water, by adding either linear or non-linear concentrationsof MDA to aliquots of the solvent. Similarly, urine can also be used toprepare a MDA concentration series by adding varying MDA concentrationsto individual urine aliquots, again recognizing that the originalconcentration of MDA in the urine is unknown and, thus, the finalconcentration is not known with complete precision.

[0070] If urine is used as the solvent for the above-described controlcolor scales, the final concentration of MDA is not precisely knownbecause the urine contains an unknown variable of MDA concentration.However, the effect of this imprecision is minimized by realizing that,given a linear correlation between the concentration of MDA and acertain color intensity generated by reacting MDA with a particularreagent (e.g., Example 1), one of ordinary skill in the art candetermine the color intensity change per unit value of MDA. By way offurther explanation, assume a baseline color, A, attributable to the MDAin the pure urine solvent. The reaction color attributable to that urinewhen supplemented by a known concentration of MDA gives a value X. Theformula (X−A)/[concentration of MDA added] is equal to the colorintensity change per unit value of MDA. Using this formula, one cancontrol for the color contribution attributable to the added MDA,thereby determining the concentration of MDA in the urine.

[0071] Reagent and acid are then added to each member of the various MDAconcentration series described above and reactions are allowed toproceed until reaction product of stable color is observed. The colorsof these reaction products are due to both the initial color of thereaction mixture and the color attributable to the MDA therein. Theresulting colors are then associated with particular concentrations ofMDA in solutions of particular initial yellow intensities. One ofordinary skill in the art will recognize that the initial colorations ofthe reaction mixtures may be due to the natural colorations ofparticular urines, to modifications to the reaction mixture colorationsby adding e.g., yellow dyes, by detecting the color of such reactionmixtures through color filters (e.g., yellow filters), or by using anymeans known in the art to approximate the various colorations of urine.

[0072] Morning urine samples from 61 healthy volunteers were firstexamined for their redox status, exactly the same procedure as inExample 1, Variant 2, being adopted. The test was carried out on thesame morning of sampling. The following frequency of the individual loadstages was recorded: I: 41 subjects II: 15 subjects III:  5 subjects

[0073] Moreover, the samples were tested for their pH value and ascorbicacid content using suitable, commercially available test strips, nocorrelation being established between these values and the redox statusdetermined according to the invention.

[0074] A particular oxidative load exhibited by an individual can beeither low, medium, or high. These oxidative loads correlate to specificconcentration ranges of relevant substances (e.g., MDA) in urine. A lowoxidative load (I) corresponds to a MDA concentration in urine rangingfrom greater than 0 to 0.5 μM. A medium oxidative load (II) correspondsto a MDA concentration in urine ranging from greater than 0.5 μM to 1.5μM. A high oxidative load (III) corresponds to a MDA concentration inurine greater than 1.5 μM up to 3.0 μM or beyond.

[0075] Three subjects whose samples exhibited a high oxidative load(Stage III) in the first test were then administered a dose of acommercially available micro-nutrient preparation on the same day (day1), after the first urine sampling, the preparation having the followinganti-oxidatively acting components: Vitamin A: 2500 I.E. = 0.75 gVitamin C: 950 mg Vitamin E: 150 mg Beta-carotin:  15 mg

[0076] and the following trace elements required for the anti-oxidativeaction of different enzymes: Selenium: 50 μg Iron: 0.8 mg Zinc: 10 mgManganese: 2 mg Copper: 0.5 mg.

[0077] On the morning of the following days (day 2 to day 4) urinesamples were again taken from the three subjects, and examined as on thefirst day. However, the administration of the micro-nutrient preparationwas not repeated on days 2 to 4. The results of the tests on these urinesamples are indicated in the following Table 3. TABLE 3 Subject Day 1Day 2 Day 3 Day 4 1 III I II III 2 III I II II 3 III II III III

[0078] This test, particularly the low load established on the first dayafter administration of the anti-oxidatively acting preparation (Day 2),clearly shows that the kit or method according to the invention aresuitable for evaluating the redox status.

1. A kit for determining redox status in an organism, comprising: (a) atleast one reagent selected from the group of compounds with the generalformula (I)

 wherein R¹ is H or C₁-C₁₀-alkyl;  and wherein R² is independentlyselected from the group consisting of H; C1-C10-alkyl; NH₂; NHR; NR₂;NHCOR; OH; OR; OCOR; SH; SR; F; Cl; Br; CF₃; or CCl₃, and wherein each Ris selected from the group consisting of C₁-C₆-alkyl;  and wherein R³and R⁴ are independently selected from the group consisting of H;C1-C10-alkyl; NH₂; NHR; NR₂; NHCOR; OH; OR; OCOR; SH; SR; F; Cl; Br;CF3; CCl₃; C₆-C₁₄-aryl; C₆-C₁₄-aryl, wherein the aryl group issubstituted by one or more substituents selected from the groupconsisting of C₁-C₆-alkyl, NH2, NHR, NR₂, NHCOR, OH, OR, OCOR, SH, SR,F, Cl, Br, CF₃ and CCl₃, and wherein each R is selected from the groupconsisting of C₁-C₆-alkyl; (b) at least one acid; and (c) a color scalefor visual color comparison, where a certain redox status of theorganism to be analyzed is determinable from individual colors on thescale.
 2. The kit according to claim 1, wherein the reagent is 2-methylindole or 1,2-dimethyl indole.
 3. The kit according to claim 1, whereinthe acid is hydrochloric acid.
 4. The kit according to claim 1, whereinthe reagent is present in the form of a solution in at least onesolvent.
 5. The kit according to claim 4, wherein the concentration ofthe reagent in the solution is 0.1 to 50 mmol/l.
 6. The kit according toclaim 1, wherein the acid is present in the form of a solution in atleast one solvent.
 7. The kit according to claim 6, wherein theconcentration of the acid in solution is 0.1 to 5.0 mol/l.
 8. The kitaccording to claim 6, wherein the solvent comprises isopropanol.
 9. Thekit according to claim 1, wherein the reagent or a solution thereof iscontained in a sealed container, which can be destroyed or opened bydeliberate mechanical action, the reagent or a solution thereof isseparate from the acid, and the reagent or solution thereof is arrangedso that the reagent or solution thereof is capable of directlycontacting the acid or a solution thereof and the urine sample as aresult of the destruction or opening of the container.
 10. The kitaccording to claim 1, wherein the acid or a solution thereof iscontained in a sealed container, which can be destroyed or opened bydeliberate mechanical action, the acid or a solution thereof is separatefrom the reagent, and the acid or a solution thereof is arranged so thatthe acid or solution thereof is capable of directly contacting thereagent or a solution thereof and the urine sample as a result of thedestruction or opening of the container.
 11. The kit according to claim1, wherein the reagent is present in solid form.
 12. The kit accordingto claim 12, wherein the reagent is present in the form of a mixturewith a neutral carrier substance.
 13. The kit according to claim 1,further comprising at least one of the following elements: a vessel forimmediate receiving of the excreted urine and an analysis vessel inwhich a urine sample, the reagent and acid are brought into contact. 14.The kit according to claim 1, further comprising an analysis vessel inwhich the urine sample, the reagent, and the acid are brought intocontact, wherein the color scale is connected to the analysis vessel sothat the color comparison between the sample solution and the colorscale can be carried out immediately and visually.
 15. The kit accordingto claim 1, wherein the reagent and the acid are both incorporated intothe test strip in solid form, wherein said test strip can be immerseddirectly in the sample to be analyzed.
 16. The kit according to claim15, wherein the acid is citric acid, oxalic acid, or trichloroaceticacid.
 17. A method for determining redox status in an organism,comprising the following steps: (a) mixing a urine sample to be analyzedwith (i) at least one reagent selected from the group of compounds withthe general formula (I)

 wherein R¹ is H or C₁-C₁₀-alkyl;  and wherein R² is independentlyselected from the group consisting of H; C₁-C₁₀-alkyl; NH₂; NHR; NR₂;NHCOR; OH; OR; OCOR; SH; SR; F; Cl; Br; CF₃; or CCl₃, and wherein each Ris selected from the group consisting of C₁-C₆-alkyl;  and wherein R³and R⁴ are independently selected from the group consisting of H;C₁-C₁₀-alkyl; NH₂; NHR; NR₂; NHCOR; OH; OR; OCOR; SH; SR; F; Cl; Br;CF₃; CCl₃; C₆-C₁₄-aryl; C₆-C₁₄-aryl, wherein the aryl group issubstituted by one or more substituents selected from the groupconsisting of C₁-C₆-alkyl, NH₂, NHR, NR₂, NHCOR, OH, OR, OCOR, SH, SR,F, Cl, Br, CF₃ and CCl₃, and wherein each R is selected from the groupconsisting of C₁-C₆-alkyl; (ii) at least one acid; and (b) visuallycomparing the coloration of the sample with a color scale, therebydetermining a specific redox status of the organism.
 18. The methodaccording to claim 17, wherein the reagent is 2-methyl indole or1,2-dimethyl indole.
 19. The method according to claim 17, wherein theacid is hydrochloric acid.
 20. The method according to claim 17, whereinthe reagent is used in the form of a solution in at least one solvent.21. The method according to claim 20, wherein the concentration of thereagent in the solution is 0.1 to 50 mmol/l.
 22. The method according toclaim 17, wherein the acid is used in the form of a solution in at leastone solvent.
 23. The method according to claim 22, wherein theconcentration of the acid in the solution is 0.1 to 5.0 mol/l.
 24. Themethod according to claim 22, wherein the solvent comprises isopropanol.25. The method according to claim 17, wherein the reagent or a solutionthereof is first contained in a sealed container, which can be destroyedor opened by deliberate mechanical action, the reagent or a solutionthereof is separate from the acid, and the reagent or solution thereofis arranged so that the reagent or solution thereof is capable ofdirectly contacting the acid or a solution thereof and the urine sampleas a result of the destruction or opening of the container.
 26. Themethod according to claim 17, wherein the acid or a solution thereof iscontained in a sealed container, which can be destroyed or opened bydeliberate mechanical action, the acid or a solution thereof is separatefrom the reagent, and the acid or a solution thereof is arranged so thatthe acid or solution thereof is capable of directly contacting thereagent or a solution thereof and the urine sample as a result of thedestruction or opening of the container.
 27. The method according toclaim 17, wherein the reagent is used in a solid form.
 28. The methodaccording to claim 27, wherein the reagent is used in the form of amixture with a neutral carrier substance.
 29. The method according toclaim 17, wherein the reagent and the acid are both used in solid formin a test strip which is immersed directly in the sample to be analyzed.30. The method according to claim 29, wherein the acid is citric acid,oxalic acid, or trichloroacetic acid.
 31. The kit according to claim 4,wherein the solvent comprises isopropanol.
 32. The method according toclaim 20, wherein the solvent comprises isopropanol.